Recent studies have provided evidence that colorectal cancer initiating cells (CCIC) are important in colorectal cancer (CRC) tumorigenesis and metastasis. In Preliminary Studies, we derived multiple new CCIC lines from both primary CRCs and metastases. We show that CCIC have significantly elevated NOTCH signaling levels vs. commonly used CRC cell lines, and that CCIC undergo cell cycle arrest and apoptosis when NOTCH is inhibited. The overall goal of this proposal is to understand the mechanism of NOTCH signaling in colon cancer initiating cells and the role of NOTCH in human colon cancer. This goal is significant because identifying critical drug targets and biomarkers for CCIC is an innovative approach to improve current CRC chemoprevention and chemotherapy. We therefore propose to (1) Identify critical NOTCH pathway components in CCIC anti- apoptosis, xenograft formation and self-renewal and (2) Test the hypothesis that Non-Steroidal Anti- Inflammatory Drugs (NSAIDs) inhibit 3-secretase activity and NOTCH signaling in Colon Cancer Initiating Cells. PUBLIC HEALTH RELEVANCE: Recent studies have provided evidence that colorectal cancer initiating cells (CCIC) are important in colorectal cancer (CRC) tumorigenesis and metastasis. In Preliminary Studies, we derived multiple new CCIC lines from both primary CRCs and metastases. We show that CCIC have significantly elevated NOTCH signaling levels vs. commonly used CRC cell lines, and that CCIC undergo cell cycle arrest and apoptosis when NOTCH is inhibited. The overall goal of this proposal is to understand the mechanism of NOTCH signaling in colon cancer initiating cells and the role of NOTCH in human colon cancer. Understanding the mechanism of NOTCH signaling in CCIC has the potential to identify more effective drug targets for CRC chemoprevention and chemotherapy and identify biomarkers directly involved in the mechanisms of tumorigenesis and metastasis.